(-)-Epicatechin does not sensitize normal cells to radiation.

<p>(A) 20 µM (-)-Epicatechin does not sensitize HNF cells to radiation (REF = 0.9). (B) (-)-Epicatechin alone does not inhibit colony formation in HNF (p>0.05).</p

(-)-Epicatechin inhibits p21 expression and does not stimulate checkpoint kinase protein 2 phosphorylation at threonine 68 in HNF cells.

<p>(A) (-)-Epicatechin inhibits p21 expression. Protein levels in 20 µM (-)-epicatechin treated samples were normalized to samples treated with 6 Gy, and *p<0.05. (B) 20 µM (-)-epicatechin does not stimulate Chk2 phosphorylation. Protein levels in (-)-epicatechin treated samples were normalized to samples that were treated with 6 Gy.</p

(-)-Epicatechin stimulates caspase 3 cleavage and expression in Panc-1 cells.

<p>Panc-1 cells were exposed to 0 or 20 µM (-)-epicatechin for 1 hour, then exposed to 6 Gy of radiation, and incubated for 72 hours. (-)-Epicatechin stimulates pro-caspase 3 expression and cleavage.</p

The effect of (-)-epicatechin on ETC protein expression in combination with radiation.

<p>(A) A representative blot for ETC protein expression in Panc-1 cells that were treated with 0–200 µM (-)-epicatechin and 0 or 6 Gy. (B) (-)-Epicatechin alone does not significantly change ETC protein expression. Protein levels are normalized to the untreated control. (B) (-)-Epicatechin induced minimal changes in ETC protein expression when combined with 6 Gy. Values were normalized to 6 Gy alone, and * p<0.05 relative to 6 Gy/0 µM (-)-epicatechin.</p

(-)-Epicatechin sensitizes cancer cells to radiation.

<p>(A) (-)-Epicatechin sensitizes Panc-1 cells to radiation with an average REF = 1.7 (B) (-)-Epicatechin sensitizes U87 cells to radiation with an average REF of 1.5. (C) (-)-Epicatechin sensitizes MIA PaCa-2 cells to radiation with an average REF = 1.2.</p

(-)-Epicatechin is a flavanol that selectively stimulates mitochondrial respiration in pancreatic cancer cells.

<p>(A) Oxygen consumption by cytochrome c oxidase (COX) from Panc-1 cells exposed to different (-)-epicatechin concentrations. Turnover is defined as consumed O₂ (nM)/(min · total protein (mg)). Respiration rates are increased by (-)-epicatechin in a concentration-dependent manner. *p<0.05, compared to 0 µM (-)-epicatechin. (B) Oxygen consumption by cytochrome c oxidase (COX) from human normal fibroblast (HNF) cells exposed to 0 or 100 µM (-)-epicatechin. Respiration rates are not increased by 100 µM (-)-epicatechin.</p

(-)-Epicatechin stimulates checkpoint kinase protein 2 phosphorylation at threonine 68 and p21 expression in Panc-1 cells.

<p>(A) (-)-Epicatechin stimulates p21 expression. Protein levels in 20 µM (-)-epicatechin treated samples were normalized to samples treated with 6 Gy. (B) 20 µM (-)-epicatechin stimulates Chk2 phosphorylation. Protein levels in (-)-epicatechin treated samples were normalized to samples that were treated with 6 Gy, and *p<0.05.</p

Cytochrome <i>c</i> release is inhibited by insulin administration.

<p>(A) Sham operated control animals (Sham, n = 3) show faint detectable amounts of Cyt<i>c</i> in the cytosolic fraction of hippocampal CA1 neurons. After 8 min of ischemia followed by 24 h of reperfusion (R24, n = 5) cytosolic Cyt<i>c</i> increases ∼4 fold (p < 0.05) and there is a trend toward mitochondrial Cyt<i>c</i> decrease (p = 0.08), both indicative of mitochondrial Cyt<i>c</i> release. Animals exposed to 8 min of ischemia followed by 24 h of reperfusion with a single bolus of IV insulin at the onset of reperfusion (T24, n = 5) demonstrate a reduction of cytosolic Cyt<i>c</i> (p < 0.05) and increased mitochondrial Cyt<i>c</i> compared to the R24 controls. (B and C) Densitometric analyses of Western blot data (Mean +/−SEM, *p < 0.05).</p

In vivo induction of cytochrome <i>c</i> Tyr97 phosphorylation in rat brain by insulin treatment.

<p>(A) Cyt<i>c</i> was isolated from rat brain after global brain ischemia (lane 1), from untreated control rats (lane 2), from sham-operated animals after insulin treatment (lane 3), and from rat brain after ischemia with insulin treatment (lane 4). Top, Western blot with an anti-phosphotyrosine antibody (4G10) reveals no detectable tyrosine phosphorylation of brain Cyt<i>c</i> under control conditions and after global brain ischemia (lanes 2 and 1, respectively), whereas tyrosine phosphorylation of Cyt<i>c</i> is strongly induced after insulin treatment (lane 3), but slightly reduced by ischemic stress (lane 4). Bottom, Coomassie gel shows equal loading (1 µg per lane) and purity of the isolated Cyt<i>c</i> species. (B) Nano-LC/ESI-MS/MS analysis of rat brain Cyt<i>c</i> after insulin treatment (corresponding to lane 3 in Fig. 5A) unambiguously identifies Tyr97 phosphorylation by fragment ions y3, y4, y5, and y6. The sequence of the peptide was definitively assigned by b3, b4, y2, y3, y4, y5, and y6. (C) Nano-LC/ESI-MS/MS analysis of rat brain Cyt<i>c</i> after global brain ischemia and insulin treatment (corresponding to lane 4 in Fig. 5A) unambiguously identifies Tyr97 phosphorylation by fragment ions y3, y4, y6, and y7. The sequence of the peptide was definitively assigned by b3, b4, y3, y4, y6, and y7.</p

Insulin prevents neuronal cell death in the CA1 hippocampus following brain ischemia.

<p>Cresyl violet stained sections (top row) show CA1 hippocampus densely populated with pyramidal neurons in sham-operated controls (Sham), and triple-label immunofluorescence (bottom row) shows these cells to be NeuN-positive (red). After 8 min of global brain ischemia followed by 14 days of reperfusion (I/R, n = 5) there is a 90% loss of CA1 neurons and an increase in Iba-1-positive microglia and GFAP-positive astrocytes (green and magenta, respectively). Animals exposed to 8 min of ischemia followed by 14 days of reperfusion with a single bolus of insulin given at the onset of reperfusion (I/R + Ins, n = 4) demonstrate a 49% increase in NeuN-positive neurons (p < 0.05) while Iba-1 and GFAP-positive cells remain unchanged.</p